A disrupted FOXP3 transcriptional signature underpins systemic regulatory T cell insufficiency in early pregnancy failure

Summary Regulatory T (Treg) cell defects are implicated in disorders of embryo implantation and placental development, but the origins of Treg cell dysfunction are unknown. Here, we comprehensively analyzed the phenotypes and transcriptional profile of peripheral blood Treg cells in individuals with early pregnancy failure (EPF). Compared to fertile subjects, EPF subjects had 32% fewer total Treg cells and 54% fewer CD45RA+CCR7+ naive Treg cells among CD4+ T cells, an altered Treg cell phenotype with reduced transcription factor FOXP3 and suppressive marker CTLA4 expression, and lower Treg:Th1 and Treg:Th17 ratios. RNA sequencing demonstrated an aberrant gene expression profile, with upregulation of pro-inflammatory genes including CSF2, IL4, IL17A, IL21, and IFNG in EPF Treg cells. In silico analysis revealed 25% of the Treg cell dysregulated genes are targets of FOXP3. We conclude that EPF is associated with systemic Treg cell defects arising due to disrupted FOXP3 transcriptional control and loss of lineage fidelity.


INTRODUCTION
Maternal-fetal immune tolerance is critical for embryo implantation and development of a robust placenta to support healthy fetal development and on-time birth.Impaired immune tolerance is implicated in disorders of uterine receptivity to implantation that give rise to early pregnancy failure (EPF), manifesting as recurrent implantation failure and/or recurrent pregnancy loss.2][3] Recurrent pregnancy loss (also termed ''recurrent miscarriage''), defined as two or more successive spontaneous pregnancy losses, affects 1%-3% of women. 2,4Genetic abnormalities of the embryo or parents and maternal endocrine disorders are identified risk factors, but the cause is unknown in approximately 50% of cases. 1,5Recurrent implantation failure is the repeated failure of normal embryos to implant successfully and occurs in an estimated 10% of women seeking reproductive medicine treatment. 3,6There is accumulating evidence pointing to immune aberrations as a causal factor in many women who experience recurrent pregnancy loss 1,7 and recurrent implantation failure, 8 but defining the precise nature of immune lesions remains a challenge and limits clinical progress. 9ey mediators of maternal-fetal tolerance are CD4 + FOXP3 + regulatory T (Treg) cells, which accumulate in the decidualized lining of the uterus where they act during embryo implantation to inhibit excessive inflammation and suppress development of immunity to paternally inherited transplantation antigens on the embryo. 10,11Recruited Treg cells include cells generated in the periphery as well as locally in the gestational tissues, where differentiation of CD4 + T cells into Treg cells is induced by human leukocyte antigen-G (HLA-G) expressed on extravillous trophoblast cells invading the decidua. 12,13These activated Treg cells interact with other immune cells to facilitate remodeling of the uterine spiral arteries necessary for robust placental development. 14,15Their abundance and functional competence are controlled by immune-regulatory cytokines, hormones, and alloantigens of paternal origin. 116][17][18] A sufficient supply of naive Treg cells of predominantly thymic origin is required to resource the uterus, 19,20 particularly for establishing first pregnancy, in which induced Foxp3 expression in naive T cells and proliferation of recruited Foxp3 + thymic Treg cells are crucial to support acquisition of protective regulatory memory. 217][28][29] Similar changes are implicated in a range of pregnancy complications to which altered placental development contribute, including preeclampsia 30 and preterm labor. 31,329][40][41] Elevated interferon gamma (IFNG) and reduced IL10 synthesis in peripheral blood T cells indicate a systemic pro-inflammatory disposition in these conditions. 42,43A causal contribution of Treg cells to embryo implantation and placentation defects is indicated by experimental depletion of Treg cells in mice.Depending on the timing and degree of Treg cell deficiency, both implantation failure and later fetal loss can ensue. 15,44,45 lack of clarity on the immune etiologies that cause EPF and the particular significance of T cells and Treg cells have hampered development of diagnostics and therapeutic interventions for recurrent pregnancy loss and recurrent implantation failure. 9,46In previous studies that have quantified Treg cells in women with EPF, phenotyping panels are often limited in scope and there is no consensus understanding of the ontogeny of Treg cell deficiency. 7,47Not all individuals with recurrent pregnancy loss show altered Treg cells, and not all studies report consistent findings (see meta-analysis by Keller et al 7 ).This underscores the need for appropriately powered studies with sufficiently detailed phenotype analysis to enable confident ascertainment of the role of Treg cells in pregnancy loss pathophysiology.
Despite emerging understanding of Treg cell phenotypic instability arising from altered gene regulatory networks as a cause of several autoimmune and inflammatory conditions, 48 this has not been evaluated in the reproductive setting.A recent study found altered gene expression in endometrial Treg cells of women with recurrent pregnancy loss but did not determine whether Treg cell changes reflect systemic defects detectable in the peripheral blood. 29We therefore hypothesized that systemic defects in Treg cell stability are associated with Treg cell defects in EPF.To evaluate this, we undertook a detailed immunophenotyping analysis of Treg, CD4 + Tconv, and CD8 + T cell populations in women with EPF and women with proven fertility, using a multi-parameter flow cytometry panel to investigate markers of suppressive capacity, proliferation state, and memory and naive subsets.We then prepared pure populations of Treg cells and conventional CD4 + T cells (Tconv cells) by fluorescence-activated cell sorting (FACS) and profiled their gene expression by RNA sequencing (RNA-seq) and in silico bioinformatics analysis.Our data reveal new insight on previously undetected Treg cell molecular aberrations that indicate systemic Treg cell defects in this common condition.

Treg cells are fewer and have an altered phenotype in EPF
To evaluate the relationship between EPF and peripheral blood T cells, we utilized 15-color flow cytometry to analyze peripheral blood mononuclear cells (PBMCs) collected at the mid-luteal phase of the menstrual cycle from subjects with previous EPF (n = 27) or proven fertility (n = 15).EPF subjects had clinical features of recurrent pregnancy loss, recurrent implantation failure, or a combination of both (see STAR Methods section for more information, and Tables S1 and S2 for clinical details).There was no difference in EPF subjects in the proportion of CD4 + T cells within the CD3 + T cell population (Figures 1A and 1B).CD8 + T cells were 16% fewer among CD3 + T cells in the EPF group compared to fertile subjects (Figure 1C, p = 0.028), but the shift in CD4 + :CD8 + ratio did not reach statistical significance (Figure 1D, p = 0.087).EPF subjects had a 61% increase in double-negative (CD4 À CD8 À ) T cells (Figure 1E, p = 0.001) and a 43% decrease in double-positive (CD4 + CD8 + ) T cells (Figure 1F, p = 0.016) compared to fertile subjects.
There were substantial differences in Treg cell numbers in EPF subjects.Within the CD4 + T cells, the proportion of FOXP3 + CD25 + CD127 -/lo Treg cells was 32% lower in the EPF group (Figures 1G and 1H, p = 0.005).The mean fluorescent intensity (MFI) of the signature Treg cell transcription factor FOXP3 in FOXP3 + Treg cells was 20% less in EPF than in fertile subjects (Figure 1I, p = 0.027).
The phenotype of Treg cells in EPF subjects was also different.Helios, a marker of FOXP3 expression stability and Treg cell suppressive capacity, 49 was unchanged in Treg cells of EPF subjects in terms of proportion of Treg cells, or MFI (Figures 2A and 2B), although the proportion of Helios + Treg cells within the whole CD4 + T cell population was reduced by 34% (Figure 2C, p = 0.006).The proportion of Treg cells expressing CTLA4, a marker of suppressive capacity, was reduced by 21% in the EPF group (Figure 2D, p = 0.019).There was no difference in CTLA4 MFI within CTLA4 + Treg cells (Figure 2E).EPF Treg cells expressed higher levels of the proliferation marker Ki67, with a 2.1-fold increase in Ki67 + Treg cells compared to Treg cells of fertile subjects (Figure 2F, p < 0.001).Altered phenotype parameters showed relationships with Treg cell frequency (Figure S1); for example, elevated Ki67 was particularly evident in EPF subjects with low Treg cells (Figure S1A).
Similar effects on Treg cell number and phenotype were seen when subgroups of EPF subjects that met strict diagnostic criteria for recurrent pregnancy loss and recurrent implantation failure were considered.Treg cells were reduced by 34% and 31% among CD4 + T cells in women with recurrent pregnancy loss and recurrent implantation failure, respectively (Figure S2A both p < 0.01).Elevated Ki67 (Figure S2D) and elevated HLADR (Figure S2F, both p < 0.05) were detected in both subgroups, while reduced CTLA4 was evident in the recurrent implantation failure but not the recurrent pregnancy loss subgroup (Figure S2C).
To assess the memory and naive phenotypes of Treg cells, CCR7 and CD45RA were evaluated.Treg cells can be defined as naive (Treg naive , CCR7 + CD45RA + ), central memory (Treg CM , CCR7 + CD45RA À ), effector memory (Treg EM , CCR7 À CD45RA À ), or terminally differentiated effector memory expressing CD45RA (Treg EMRA, CCR7 À CD45RA + ). 52Within the total Treg cell pool of EPF subjects there was a 28% decrease in the proportion of Treg naive cells and a 34% increase in the proportion of Treg CM cells, compared to fertile subjects (Figure 2K, both p < 0.05), while Treg EM and Treg EMRA were comparable (Figure 2K).Because of the substantial reduction in Treg cells among CD4 + cells, this corresponds to a 54% reduction in Treg naive cells, while Treg CM were unchanged as a fraction of CD4 + cells (Figures 2L and 2M).Comparable reductions in Treg naive as a fraction of CD4 + T cells were also evident in subject subgroups meeting diagnostic criteria for recurrent pregnancy loss (52%, p = 0.019) and recurrent implantation failure (55%, p = 0.016) (Figure S2I).When Ki67 was evaluated as a function of memory phenotype, Treg naive and Treg EMRA were substantially more proliferative in EPF than in fertile subjects, while Treg CM and Treg EM were similar (Figure S3).
Collectively, the flow cytometry analysis revealed reduced abundance of CD4 + Treg cells in EPF subjects, with lower FOXP3 and CTLA4 expression, but retention of a HLADR hi subset.Elevated Ki67 expression indicated a greater proportion of Treg cells were proliferating in EPF subjects, and analysis of memory phenotypes showed the deficit was associated with fewer naive Treg cells among the Treg cell pool.

CD4 + Tconv cells have a pro-inflammatory shift in EPF
We then assessed the activation phenotype of the CD4 + Tconv cells of subjects with EPF or proven fertility, by measuring expression of Tbet, the Th1 cell-defining transcription factor, and RORgt, the Th17 cell-defining transcription factor.A greater proportion of CD4 + Tconv cells expressed Tbet in EPF patients (Figure 3A, p = 0.048).Because of fewer Treg cells and more Th1 cells, the ratio of Treg:Th1 cells was decreased in EPF subjects by 57% (Figure 3B, p = 0.023).The percentage of Tconv cells expressing RORgt was elevated by 1.9-fold in EPF subjects (Figure 3C, p = 0.013), causing a 74% decline in the Treg:Th17 cell ratio (Figure 3D, p < 0.027).These parameters indicate a pro-inflammatory shift in CD4 + Tconv cells in EPF subjects.The proportion of CD4 + Tconv cells expressing Ki67 was similar in fertile and EPF subjects (Figure 3E), and their memory phenotype distribution was also comparable (Figure 3F).A reduced Treg:Th1 ratio was evident in both recurrent pregnancy loss and recurrent implantation failure (both p < 0.05, Figure S2K), and a reduced Treg:Th17 ratio was also evident in both groups (both p < 0.03, Figure S2L).

tSNE analysis confirms CD3 + T cell changes in EPF
t-distributed stochastic neighbor embedding (tSNE) utilizes an unsupervised, unbiased algorithm to visualize data using all marker information. 53Unbiased X-shift clustering 54 using 7,344 CD3 + T cells from each participant identified 16 separate cell clusters among which CD4 + Treg cells, CD4 + Tconv cells, CD8 + Tconv cells, double-negative CD4 À CD8 À T cells, and double-positive CD4 + CD8 + T cell subsets were identified (Figure 4A).The MFI of 13 cell markers was determined for each of the 16 clusters (Figure 4B), and expression of each marker was mapped onto individual tSNE plots (Figure S4).
The tSNE and X-Shift analysis of Treg cells confirmed aspects of the conventional 2D analysis.The proportions of 3 of the 8 clusters were altered in the EPF samples.Cluster 8, comprising Treg cells with strong expression of FOXP3, CD25, and CTLA4 and variable expression of HLADR and Ki67 (Figure 5B) indicating robust, highly suppressive, proliferating Treg cells, was reduced in the EPF group compared to the proven fertile controls (Figure 5C).Cluster 3 containing primarily CCR7 À CD45RA + Treg EMRA cells with lower expression of FOXP3, CD25, HLADR, CTLA4, and Ki67 (Figure 5B) was markedly reduced in the EPF subjects (Figure 5C).This was counterbalanced by an increase in Cluster 4, containing mainly CCR7 + CD45RA À Treg CM cells (Figure 5B), in EPF subjects (Figure 5C).The clusters that contained mostly CCR7 + CD45RA + Treg naive cells, Clusters 1 and 6 (Figure 5B), were not different in EPF subjects (Figure 5C), but since Treg naive cells did not cluster cleanly from other Treg cells, this analysis did not provide conclusive information on Treg naive cells.

RNA-seq in Treg and Tconv cells in EPF
To evaluate whether T cell gene expression changes contribute to the altered T cell profile seen in EPF subjects, we isolated Treg and Tconv cells from cryopreserved PBMCs using FACS, stimulated the cells in culture, and then measured transcription by RNA-seq (Figure 6A).RNAseq was performed for 27 Tconv cell samples (n = 11 for fertile and n = 16 for EPF) and 15 Treg cell samples (n = 7 for fertile and n = 8 for EPF).Due to low numbers of Treg cells causing a limited cell yield after FACS, there were fewer RNA-seq samples for Treg cells than Tconv cells.Using minimum expression criteria of R1 count per million (cpm) reads in at least 6 samples, the T cells were seen to express a total of 13,175 genes.The largest driver of variation in the data identified by principal-component analysis (PCA) was T cell type, causing Treg and Tconv samples to cluster separately on PC1 (Figure 6B).
Comparison of the differentially expressed (DE) genes between Treg and Tconv cells from EPF and fertile subjects revealed significant differences.In fertile subjects, 6,656 total DE genes, or 399 with a log-fold change (lfc) >1, equivalent to a 2-fold difference in expression, were identified between Treg cells and Tconv cells (Figure S5A).Among the most substantially upregulated DE genes were FOXP3, IKZF2 (Helios), TNFSF1B (TNFR2), CCR8, TOX, CTLA4, IL1R1, and IL1R2 and downregulated DE genes were IL7R and IRF8 (Figure S5B), as expected. 29A similar number of 6,634 DE genes, or 396 with lfc >1, were identified for EPF Treg cells compared to EPF Tconv cells (Figure S5C).As in the fertile group, the DE genes included FOXP3, IKZF2, CTLA4, and IL7R (Figure S5D).However, only 64.4% of the genes DE in Treg versus Tconv cells overlapped between the fertile and EPF subjects (Figure S5E).To further investigate differences associated with fertility in the T cells, we proceeded to analyze the cell types separately.

DE genes in Tconv cells in EPF
Three Tconv samples from the EPF group were excluded from the RNA-seq analysis when PCA showed they failed to cluster within the 95% confidence interval (CI) for the group (Figure S6A).After this, PCA showed overlapping clusters of fertile Tconv and EPF Tconv cells (Figure 6C).Differential gene expression (DGE) analysis was then performed with limma/voom, 55 and, since cDNA libraries were prepared in batches, this was factored into the model to account for batch variation.780 DE genes were identified in Tconv cells from the EPF group compared with proven fertile controls.Of these, most of the genes were downregulated (719 genes) whereas 61 genes were upregulated in EPF Tconv cells (Figures 6D and S6B).
Given our finding of increased pro-inflammatory Tconv cell responses in EPF subjects, the DE genes were screened for their association with inflammation or differentiation to specific Th subsets.The chemokine receptor CCR4 is associated with Th2 cells and was increased in EPF Tconv cells.While IL17RA, a marker of inflammation, was increased in Tconv cells from EPF subjects, overall there was little evidence of increased pro-inflammatory function in the Tconv cell compartment in EPF subjects compared to fertile controls.In fact, some genes pointed to less effector activity, for example, decreased expression of the transcription factors RUNX1, RUNX2, and RUNX3 (Table S3).
Minimal disruption to genes associated with T cell effector functions was mirrored in the DE gene pathway analysis for EPF Tconv cells.Over-representation analysis was performed to identify enrichment for gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms in the significantly DE gene sets for Tconv.The full list of Tconv DE genes yielded no significantly enriched GO or KEGG terms, so a log-fold change cutoff of 0.04 (equivalent to 1.03-fold difference in expression) was applied to narrow the results list to 241 DE genes for pathway analysis (Figure S6C).Tconv DE genes for EPF then showed enrichment for two GO terms, histone methyltransferase complex and histone H3-K4 dimethylation (Figure S6D).
To widen screening for patterns in the gene expression data, we employed gene set enrichment analysis (GSEA) using the Molecular Signatures Database (MSigDB) (Table S4).Some significantly enriched pathways were identified by GSEA, including upregulation of genes in the cell cycle pathways Kong E2F3 targets and Zhou cell cycle genes in immune response at 6 h.Additionally, pathways from the C7 Immune Signatures and C3 MicroRNA Targets (MIR) legacy databases were negatively enriched, indicating genes associated with those pathways were downregulated in EPF Tconv cells.Immune Signatures pathway analysis showed suppression of genes known to be upregulated in PBMCs after YF17D vaccination (GSE13485) or downregulated in PBMCs after influenza vaccination (GSE29617).Overall, there was little evidence for inflammatory dysfunction in the Tconv cells from EPF subjects.

DE genes in Treg cells in EPF
Initial PCA of Treg cell samples did not show clustering driven by fertility, and few significantly DE genes were identified in a first-pass limma/ voom analysis (Figure S7A).Therefore we performed normalization using RUV (Remove Unwanted Variation) methodology to improve the detection of DE genes. 56For this, ''in silico empirical'' negative controls were formed using all but the top 5,000 genes ranked by edgeR p values, representing the least significantly DE genes.After RUV normalization together with exclusion of one proven fertile sample that failed to cluster with the rest of the group, Treg cell samples were seen to cluster by fertility status in the PCA plot (Figure 6E).Using the edgeR negative binomial generalized linear model (GLM) approach, 234 genes were identified as DE between Treg cells from the EPF and fertile groups, with 80 of these genes downregulated and 154 genes upregulated in Treg cells from the EPF group (Figures 6F and 7A; Table S5).
The expression level of selected genes that were significantly DE in Treg cells from EPF versus fertile subjects is shown for each sample in a heatmap (Figure 7C).Interestingly, 9 of the top 20 DE genes in Treg cells from the EPF group were cytokine genes with increased expression.Many of these cytokines are hallmark pro-inflammatory factors normally associated with highly activated and differentiated T helper cells, including IFNG, IL4, IL17A, and CSF2.This suggests Treg cells have compromised linage stability in EPF subjects.To investigate this, FOXP3 expression was examined, and a trend to downregulated FOXP3 expression in Treg cells from EPF compared to fertile subjects was found (Figure S8A, p = 0.108), consistent with the finding of reduced FOXP3 protein by flow cytometry (Figure 2C).The list of Treg cell DE genes was then screened for FOXP3 chromatin immunoprecipitation (ChIP) targets. 57Of the 234 DE genes in EPF Treg cells, 60 (25.6%) were FOXP3 ChIP hits, suggesting Treg cell gene dysregulation in EPF subjects is at least partly due to reduced FOXP3 (annotated in Table S5).
Other DE transcription factors in EPF Treg cells included upregulated estrogen receptor 2 (ESR2), SRY-box transcription factor 13 (SOX13), interferon regulatory factor 7 (IRF7), IRF9, and Kruppel-like factor 4 (KLF4) and downregulated transcription factor 7 (TCF7, encoding TCF1).Some of these changes would be expected to benefit Treg cell function, such as increased ESR2, as estrogen signaling enhances Treg cell expansion. 58Others, like IRF7, can have dual effects or negatively affect Treg cell function, like IRF9 and KLF4.While transcription factor IRF7 binds to the FOXP3 promoter and drives FOXP3 expression, 59 both IRF7 and IRF9 are positive regulators of type I interferon genes. 60,61KLF4, on the other hand, regulates IL17 expression during Th17 differentiation. 62ysregulation of both pro-and anti-Treg cell gene expression within Treg cells of EPF subjects is evident.Notably pro-inflammatory gene TNF was upregulated, as were anti-inflammatory genes including IL10 and CTLA4 and chemokines CCL3 and CCL4, which are implicated in guiding CCR5 + effector CD4 + and CD8 + T cells toward Treg cells to enable their suppression. 63CXCR5 and PDCD1 (encoding PD-1), which are expressed by T follicular regulatory (Tfr) cells, were also upregulated.Although this might indicate increased Tfr differentiation, other genes normally associated with a Tfr signature such as BCL6 and ICOS were not DE.

Treg cell pathways implicated in EPF
Among the Treg cell DE genes, over-representation analysis revealed enrichment of 15 GO terms (Table S6) and 5 KEGG terms (Table S7).The most significantly over-represented GO term was related to the immune system, hematopoietic or lymphoid organ development.Other immune system-related GO terms for Treg cells included 3 related to cell movement (regulation of locomotion, regulation of cell migration, myeloid leukocyte migration) and 6 related to cell signaling (signaling receptor regulator activity, receptor ligand activity, G protein-coupled receptor signaling pathway, positive regulation of tyrosine phosphorylation of STAT protein), including 2 terms related to cytokine production and signaling (cytokine production, cytokine receptor binding) (Figure 7B).Terms in the cellular component ontology of GO terms were all related to the cell surface (integral component of plasma membrane, plasma membrane region, cell surface, apical part of cell).
GSEA was also performed for the Treg cell DE genes (for a full list of enriched terms, see Table S8).Increased T cell activation was inferred from the positive enrichment of KEGG T cell receptor signaling pathway and primary immunodeficiency (PID) T cell receptor (TCR) calcium pathway from MSigDB.The PID IL23 pathway was positively enriched, as was hallmark TNFA signaling via NFKB, providing further evidence for aberrant pro-inflammatory cytokine pathway activation in EPF Treg cells.Other enriched cytokine signaling pathways were reactome interleukin-2 family signaling, marzec IL2 signaling up, reactome interleukin 10 signaling, KEGG cytokine-cytokine receptor interaction, and KEGG JAK STAT signaling pathway.There was negative enrichment of many GSEA pathways for Treg cells from EPF subjects associated with cell cycle progression or proliferation, indicating downregulation of cell cycle-related genes.Enriched pathways associated with cell cycle included Hallmark E2F targets, Hallmark G2M checkpoint, Ishida E2F targets, Kong E2F3 targets, Fischer G2 M cell cycle, Zhou cell cycle genes in immune response at 6 h (and 24 h), and Graham CML dividing vs. normal quiescent up (Table S8).
We also investigated whether the genetic regions of the Treg cell DE genes were associated with particular diseases by performing enrichment analysis against the 2019 genome-wide association study (GWAS) database using the web-based tool Enrichr.Significant enrichment was identified for Celiac disease, Type 1 diabetes, and Itch intensity from mosquito bite (Table S9).Among the DE genes associated with these conditions were genes related to Treg cell regulatory function (IL10, CTLA4, THEMIS), negative regulation of TCR signaling (UBASH3A, CTLA4), and pro-inflammatory function (IFNG).

Treg cell changes are independent of clinical evidence of autoimmunity
The clinical guidelines of the European Society for Human Reproduction and Embryology (ESHRE) for the management of recurrent pregnancy loss 4 recommend testing for autoantibodies (antinuclear, anticardiplin, B2-glycoprotein, and lupus anticoagulant) and thyroid function.Of the 27 EPF subjects, clinical data were accessible for 25, and 9 were positive for one or more autoimmune parameters, enabling classification of autoimmune status (Table S2).Analysis of flow cytometry data did not reveal a difference in Treg cell abundance, proliferation, or activation state according to autoimmune status, nor was there any change between the Treg:Th1 or Treg:Th17 ratios (Figures 8 and S9).Similarly, there was no evidence of association between autoimmune status and Treg cell defects as measured by transcriptional analysis (Figure 7C), including FOXP3 gene expression (Figure S8).S2) are identified by hash symbol.

DISCUSSION
The degree to which a perturbed Treg cell pool contributes to EPF and the nature of the underlying defects are not well defined.Here we have used multi-colored flow cytometry and RNA-seq analysis to evaluate the prospect of a systemic shift in Treg cell phenotype.Collectively, the data reveal substantial changes in Treg cell abundance and phenotype, associated with transcriptional abnormalities indicative of impaired lineage stability and compromised immunosuppressive capacity, in a high proportion of EPF subjects.The Treg cell defects cause impaired constraint of pro-inflammatory cells indicated by increased RORgt + Th17 cells, and substantially lower Treg:Th17 and Treg:Th1 cell ratios in EPF subjects.Since T cell populations residing in the uterine decidua largely originate from the peripheral blood and reflect systemic conditions, 11,23 these changes have potential to disrupt pregnancy tolerance and impair embryo implantation.Importantly, altered Treg cell cellular and transcriptional parameters were evident in women experiencing EPF without identified autoantibodies or thyroid dysfunction, as well as those positive for indicators of autoimmunity.
The altered Treg cell profile in EPF was associated with skewing of the Treg cell population toward substantially fewer CCR7 + CD45RA + Treg naive cells.Several biologically plausible scenarios may contribute.Naive Treg cells, like other T cells, are generated in the thymus before export via the blood to circulate through the body and secondary lymphoid organs. 64Encountering antigen triggers Treg cell differentiation to effector and memory cell subsets. 65Whereas Treg CM continue to circulate, most Treg EM and Treg EMRA cells will egress to tissues to elicit their suppressive functions and die sooner than naive or central memory Treg cells. 66The causes of Treg naive cell deficit therefore include ( 1 S2).Data on autoimmune status is not available (n/a) for proven fertile women or 2/27 EPF women.Values for flow cytometry parameters from proven fertile and EPF subjects are displayed in a heatmap showing relative score in each sample based on Z scores calculated from data shown in Figures 1, 2, and 3. Higher intensity of red indicates the parameter is higher relative to mean values across all samples.See Figure S9 for additional visualization of flow cytometry data according to autoimmune status.
Our evidence strongly indicates lineage instability in Treg cells, which causes loss from the Treg cell pool during differentiation and conversion to effector T cell subsets. 48This interpretation is supported by the lower abundance of FOXP3 in Treg cells of EPF subjects, and their altered gene expression profile upon TCR stimulation.This is coupled with increased transcription of pro-inflammatory cytokines, including IFNG, TNF, IL17A, and IL21, and lower surface CTLA4 expression seen by flow cytometry.These findings concur with a previous study that reported reduced FOXP3 MFI in Treg cells, and less FOXP3 mRNA, in women with unexplained recurrent pregnancy loss. 37he interpretation of reduced Treg cell stability in EPF is supported by reduced expression of Helios, a marker of Treg cell stability, 49 in decidual Treg cells of miscarriage subjects. 27Demethylation of the Treg cell-specific demethylated region (TSDR) within the FOXP3 promotor provides stable FOXP3 expression, leading to cells with strong suppressive capacity. 67,68In mice, there is further demethylation of this site in early pregnancy, 19 implying Treg cell stability is important for pregnancy success.Increased methylation in the TSDR in unexplained miscarriage correlates with waning Treg cell abundance and suppressive capacity. 36ur data also point to a faster rate of Treg cell maturation contributing to decreased Treg cell abundance.Gene pathway analysis revealed transcriptional enrichment for TCR signaling pathways, suggesting that Treg cells from EPF women are hyper-responsive to TCR stimulation resulting in increased differentiation to effector and memory Treg cell subsets.A lower proportion of naive CD45RA + Treg cells, with increased memory phenotype, is reported in women with unsuccessful in vitro fertilization (IVF) treatment. 69A similar change in Treg cell phenotype is observed in clinical conditions where immune dysregulation and breakdown of immune tolerance are implicated including multiple sclerosis 70 and type 1 diabetes. 71,72The increase in PDCD1 mRNA (encoding PD-1, CD279) in Treg cells from EPF subjects (Table S5) supports the interpretation of accelerated maturation and exhaustion in Treg cells.This aligns with previous findings of elevated exhaustion marker PD-1 on Treg cells from EPF 39 and recurrent implantation failure 35 subjects, and the possible benefit of intravenous immunoglobulin (IVIG), an experimental therapy for recurrent pregnancy loss 73 that acts to reduce PD-1 + exhausted Treg cells in peripheral blood. 74f increased differentiation and conversion of Treg cells occur in EPF, this would increase demand on the supply of naive Treg cells.Thymic production of new Treg cells in humans declines after puberty so that in adults the peripheral pool of naive cells is maintained primarily by proliferation of mature naive (MN) Treg cells. 75Elevated Ki67 expression in EPF women might represent an attempt to maintain the pool of naive Treg cells.This is borne out in the observation that Treg cell proliferation was greatest in the EPF subjects exhibiting the lowest Treg cell abundancies (Figure S1A).Our results are reminiscent of the observation of increased proliferating Ki67 + Treg cells despite diminished Treg cells in the decidua from miscarried euploid embryos. 28There was no correlation between the number of pregnancy losses and the degree of Treg cell deficiency in EPF subjects (Figure S1G), similar to a previous report. 76n contrast to the flow cytometry data, RNA-seq GSEA analysis indicated suppression of cell cycle progression in EPF Treg cells (Table S8).This discrepancy likely reflects differential responses to in vitro stimulation of T cells prior to transcriptome analysis.In vitro activation is a standard approach to reveal the functional potential of T cells 77,78 and is appropriate given early pregnancy exposes T cells to activating stimuli, but whether it authentically replicates in vivo conditions is unclear.Since proliferation was not increased in Treg EM and Treg CM cells (Figure S3) and cell cycle pathways were actually suppressed in EPF Treg cells, EPF Treg cells are unlikely to be inherently hyper-proliferative.Instead, the data support the interpretation that naive Treg cell proliferation is increased to maintain the naive Treg cell pool.It is possible that insufficient thymic generation of new Treg cells contributes to a depleted naive Treg cell pool in EPF, and future studies to quantify recent thymic emigrants (RTE) using T cell receptor excision circles (TREC) or CD31 79 are warranted.The ratio of RTE:MN Treg cells determines the suppressive capacity of the total naive Treg cell pool 80 and reasonably might be reduced in EPF.
Alternative explanations for reduced total Treg cells in the blood of EPF subjects are increased cell death or redistribution of the Treg cells to peripheral tissues.No evidence of increased cell death was identified in the transcriptional analysis.The lack of any change in chemokine receptor expression except for increased CXCR5, a marker of T follicular regulatory cells, 81 does not support a change in egress from the blood.Nevertheless, regulation of cell migration and locomotion were enriched GO terms for EPF Treg cells, so further investigation of this possibility is warranted.
A key finding was reduced expression of CTLA4, implying that Treg cells from EPF women have impaired suppressive capacity.In fertile women, Treg cells were nearly uniformly 75%-90% CTLA4 + , while a bimodal pattern of CTLA4 expression occurred in EPF women such that CTLA4 + expression correlated inversely with Treg cell proportion (Figure S1B).CTLA4 is a negative regulator of TCR signaling that can be induced by TCR hyper-stimulation in an attempt to constrain CD28 co-stimulation. 82Stimulation of Treg cells in vitro prior to cDNA library preparation upregulates CTLA4, 83 potentially explaining the small upturn we saw in CTLA4 expression in EPF women.We had insufficient cells to conduct suppression assays, the gold standard for measuring Treg cell functional competency, but this will be a priority in follow-up studies.Others have observed reduced Treg cell suppressive capacity in decidual Treg cells 26 and peripheral blood Treg cells 22 in EPF.
The EPF women in our study had an elevated proportion of HLADR hi memory Treg cells, a population normally endowed with high suppressive capacity. 51,84EPF subjects with the fewest Treg cells tended to have the highest percentage of HLADR hi Treg cells (Figure S1C).Collectively, increased Treg cell CTLA4, HLADR, and Ki67 expression suggests a compensatory attempt to counteract reduced Treg cell frequency.However this shift may be counterproductive, as women undergoing IVF showed higher suppressive activity of HLADR + memory Treg cells when pregnancy failed. 69The possibility that Treg cells mature faster, leading to an earlier death, is supported by HLADR hi Treg cells exhibiting greater proliferation (Figure S1D), and elevated susceptibility to apoptosis. 84 comparison of DE genes in the Treg cells from EPF subjects compared to fertile subjects demonstrated that EPF Treg cells had increased pro-inflammatory IFNG, IL4, IL17A, and CSF2.This may indicate their specialized differentiation to Treg cell subsets suited for suppression of cognate T helper subsets, 85 or less phenotypic stability resulting in the adoption of pro-inflammatory signatures after stimulation, as would be expected with reduced FOXP3 protein.Treg cells from EPF subjects exhibited upregulated KLF4, a transcription factor which positively regulates IL17, using Th17 differentiation, 62,86 and upregulation of IRF7 and IRF9, key drivers of interferon gene and STAT1/STAT2 pathways.60,61,87 Reduced expression of TCF7 (encoding the TCF1 protein) in EPF Treg cells is another indication of reduced phenotype stability.TCF1 interacts with FOXP3 to cooperate in regulation of genes important for Treg cell function.88,89 Treg cell-specific deletion of Tcf7 in mice shows it restrains excessive cell cycling and maintains suppressive function and may positively regulate Foxp3 expression.89 Downregulation of TCF7 in Treg cells from EPF patients is thus likely to be detrimental.A regulatory link with SOX13, another transcription factor that was DE in EPF Treg cells, may exist.TCF1 acts as a transcriptional activator in the presence of b-catenin in canonical Wnt signaling, 88 which is suppressed by SOX13.SOX13 was upregulated in EPF Treg cells, which is likely to compound impaired TCF7 expression to exacerbate Treg cell functional incompetence.
Other transcription factors expected to benefit Treg cell function were upregulated.One example is estrogen receptor ESR2, which mediates estrogen-mediated promotion of Treg cell induction, expansion, and stability. 58Other upregulated genes are anti-inflammatory in nature.IL10 and the chemokines CCL3 and CCL4 have been implicated as a mechanism of guiding CCR5 + effector CD4 + and CD8 + T cells toward Treg cells to facilitate suppression. 63The upregulation of genes normally expected to improve Treg cell function indicates further compensatory adaptations.
There was little evidence of faults in the Tconv cell arm, and some gene changes indicate less effector activity.RUNX1, RUNX2, and RUNX3, genes involved in mature T cell function and differentiation to effector or memory subsets, 90 were all downregulated in EPF Tconv cells.The increase in Th17 cells observed in EPF subjects and consequent decrease in the Treg:Th17 ratio presumably reflect insufficient Treg cell suppression and mirror earlier findings. 35,91Although IL17RA expression was increased in EPF Tconv and IL17 cells, IL17RA-mediated effects are linked to suppressive resistance of non-Th17 effector CD4 + T cells. 92Another observation of note was reduced CD4 + CD127 hi cells in Tconv cells of EPF subjects.These cells have anti-inflammatory properties and are implicated in suppressing progression of type 1 diabetes. 93any women with recurrent pregnancy loss and recurrent implantation failure have elevated anti-phospholipid, anticardiolipin, or other autoantibodies. 94,95We found that low Treg cell numbers, reduced FOXP3 MFI, and altered Treg cell transcriptional profiles were not linked with clinical indication of autoimmunity, and EPF women with autoantibodies did not exhibit worse Treg cell features.This suggests that Treg cell phenotype and transcriptional parameters may be more sensitive indicators of impaired pregnancy tolerance than autoantibody status.
Fewer naive Treg cells with impaired suppressive competence in peripheral blood is likely to be a constraint on availability of Treg cells to supply the endometrial compartment, given endometrial Treg cells are drawn from the blood most likely as naive cells that differentiate after extravasation. 23,24][99][100][101] Nevertheless we acknowledge that specific Treg cell phenotypes are enriched in the endometrium, so that the endometrial Treg cell population is not identical to peripheral blood. 23,24,29Decidual Treg cells have a higher proportion of clonally expanded populations and enhanced suppressive capacity. 23,102A recent study reports differences in transcriptomes of Treg cells from blood and endometrium in EPF patients and indicates that endometrial Treg cells acquire a comparatively stronger regulatory profile and memory phenotype. 29An effort to identify differences in endometrial Treg cells in women with recurrent pregnancy loss revealed lower cell surface TIGIT and elevated S1PR1 gene expression but found FOXP3, HELIOS, and CTLA4 were not different. 29The decrease in TIGIT expression is particularly interesting given the recent description of 3 distinct Treg subpopulations within the decidua: 1) CD25 hi FOXP3 + , 2) PD-1 hi IL10 + , and 3) TIGIT + FOXP3 dim , all of which express high TIGIT 103 and exhibit strong potential to suppress anti-fetal immunity. 103,104Future studies should therefore include analysis of both tissue compartments.
In summary, this study indicates that Treg cell dysfunction and compromised immune tolerance can arise in EPF due to an altered Treg cell transcriptional profile that impairs Treg cell suppressive competence.Altered FOXP3 expression leading to loss of Treg cell functional fitness and stability is implicated, and this may precede or be independent of synthesis of antibodies to common autoantigens.The similarity between the transcriptional changes seen in EPF and autoimmune conditions type 1 diabetes and celiac disease indicates the possibility of shared underlying Treg cell defects.These and other autoimmune conditions all appear to arise secondary to subtle alterations to the gene regulatory networks controlled by FOXP3. 47,85,105Our findings are consistent with emerging views that Treg cell instability is caused by single nucleotide polymorphisms (SNPs) within the FOXP3 gene body, or nearby noncoding regions or target genes, interacting with genetic disposition and precipitated by environmental and developmental factors. 47Collectively, our observations bolster the evidence that at least in some women EPF is due to underlying disruption in the Treg cell compartment and provide new insight on how this can arise at a genomic level.The findings strengthen the impetus for development of intervention strategies that modulate Treg cells for application in the reproductive medicine setting. 46

Limitations of the study
The current study is limited by the small study size and the heterogeneity of the EPF patients' clinical histories, so it is not possible to extrapolate from the current findings to recurrent pregnancy loss and recurrent implantation failure more broadly.Although women meeting diagnostic criteria for recurrent pregnancy loss and recurrent implantation failure showed broadly similar Treg cell phenotypes to the EPF group as a whole (Figure S2), there were insufficient numbers for independent transcriptome analysis.The considerable between-individual variance in transcriptomic profile indicates that not all women with EPF have abnormal Treg cells, and, within those that do, a range of degrees and types of Treg cell dysfunction likely exist.A larger study is now required to determine what proportion of EPF is associated with altered Treg cell features, to investigate the heterogeneity in Treg cell phenotypes and relationships with age and clinical features, and to evaluate transcriptional differences that may distinguish different clinical categories.
Another limitation is the lack of information on genetic status of lost embryos.Around 50% of miscarriages involve embryo aneuploidy, when loss is considered a normal physiological response.Aberrant decidual Treg cells are more apparent in women with loss of normal engaged in reproductive medicine treatment cycles or taking hormones or other fertility medication in the cycle when blood was collected.Within 4 h of sample collection, blood was centrifuged at 2000 x g for 10 min at room temperature (RT), and plasma was removed.Dulbecco's modified phosphate-buffered saline (DPBS, Sigma-Aldrich, St. Louis, MO) was then added in a volume equal to the plasma removed, then further DPBS was added to the sample to double the volume, before layering over Lympholyte-H cell separation media (Cedarlane Laboratories, Burlington, Canada) and centrifugation at 600 x g for 30 min at RT without brake.The PBMC-containing layer was collected, washed twice in DPBS, centrifuged at 450 x g for 5 min and cells were resuspended in complete RPMI-1640 media (RPMI, Thermo Fisher) containing 10% FBS (Stemcell Technologies, Vancouver, Canada) and antibiotic and antimycotic (Sigma Aldrich).Cells were labeled with trypan blue (Sigma-Aldrich, St Louis, MO) and total viable cells were counted using a hemocytometer.Cell concentration was adjusted to $6 3 10 6 cells per mL in complete media, with an equal volume of freezing media (20% DMSO [Sigma-Aldrich] and 80% FBS) added dropwise.Aliquots containing $3 3 10 6 PBMCs, were stored in cryovials and frozen at a controlled rate to À80 C in a Mr Frosty (Nalgene, Rochester, NY) overnight.The following day PBMC cryovials were transferred into liquid nitrogen storage.

Flow cytometry
Cryopreserved PBMC samples were thawed, washed in complete RPMI and counted using a haemocytometer and trypan blue.Cells were rested overnight under cell culture conditions in a 96 well plate at 1 3 10 6 /mL.The following day, cells were washed in PBS and incubated with fixable viability stain 575V (1/1000 dilution) and human Fc block (1/30 dilution) in PBS for 10 min.Without washing cells, CCR7-BUV395 antibody was added and allowed to incubate for 15 min, before the addition of the surface-staining antibody master mix (CD3-APC-H7, CD4-BUV496, CD8-BUV737, CD25-BV786, CD127-PE-Cy7, CD45RA-BB515 and HLADR-V510, more details in Table S10), prepared in Brilliant Staining Buffer Plus (BD Biosciences) and a further 20 min incubation.Cells were washed in PBS and then fixed and permeabilized using the FOXP3 Transcription Factor Staining Buffer Set (Thermo Fisher) according to the manufacturer's instructions.Cells were washed in permeabilization buffer and then incubated for 40 min at RT with a master mix of intracellular-staining antibodies (FOXP3-PECF594, Helios-AF647, CTLA4-PE-Cy5, Ki67-AF700, RORgt-PE, Tbet-BV421, more details in Table S10) prepared in Brilliant Staining Buffer Plus.All incubations were performed in the dark.Cells were washed and resuspended in permeabilization buffer, and analyzed on a BD LSR Fortessa X20 flow cytometer with FACSDiva software (BD Biosciences).In each experiment fluorescence minus one (FMO) and unlabelled controls were run to assist with gating positive and negative populations and single color compensation controls were performed utilizing either BD anti-mouse Igk CompBeads (BD Biosciences) or UltraComp eBeads (Thermo Fisher).

Flow cytometry data analysis
All flow cytometry data was analyzed using FlowJo software (BD Biosciences, version 10.6.2).Gates were established to exclude dead cells, debris and doublets, and assess subpopulations of viable CD3 + T cells (gating strategy in Figure S10).2-dimensional flow cytometry plots were used to identify CD4 + , CD8 + , CD4 + CD8 + and CD4 -CD8 -T cells.Treg cells were defined as CD3 + CD4 + CD25 + FOXP3 + CD127 À/lo and were further assessed for phenotype, using Helios, CTLA4, HLADR, and CD45RA, surrogate markers for Treg cell functional capacity, and using Ki67 as a marker of proliferation.Non-Treg CD4 Tconv and CD8 Tconv populations were analyzed for Tbet and RORgt expression to identify Th1 and Th17 T cells, respectively.All T cell compartments were assessed for the expression of CCR7 and CD45RA to identify naive (T na i ve , CCR7 + CD45RA + ), central memory (T CM , CCR7 + CD45RA À ), effector memory (T EM , CCR7 À CD45RA À ) and terminally differentiated effector memory expressing CD45RA (T EMRA, CCR7 À CD45RA + ) 52 phenotypes.Data are presented as the percentage of cells expressing specific markers or the geometric mean fluorescence intensity (MFI) of an individual marker within a specified population.
Flow cytometry data were also analyzed using the nonbiased, nonlinear dimensionality reducing t-distributed stochastic neighbor embedding (tSNE) algorithm 53 and clustering algorithm, X-shift. 54Firstly, viable, singleton CD3 + T cells from the 42 samples (15 fertile and 27 EPF) were gated (Figure S10).Events with extreme fluorescence (negative or positive) were excluded.A downsample of 7,344 viable CD3 + T cells from each sample was concatenated into a single file.The tSNE was constructed in FlowJo taking into account the fluorescence of CD4, CD8, FOXP3, CD25, CD127, CCR7, CD45RA, HLADR, Helios, CTLA4, Tbet, RORgt and Ki67, using 1000 iterations of the Barnes-Hut algorithm, with a perplexity of 30 and learning rate of 1000.X-shift clustering analysis 54 was performed using expression of the same 13 markers, with the k nearest neighbor default value of 102, distance metric set at Euclidean and a 100,000 cell subsampling limit set.
A second tSNE was constructed using viable CD3 + CD4 + FOXP3 + CD25 + CD127 -/lo Treg cells.Each sample had 418 Treg cells concatenated into a single file and a tSNE plot generated using the fluorescence of CCR7, CD45RA, HLADR, Helios, CTLA4 and Ki67, using 1000 iterations of the Barnes-Hut algorithm, with a perplexity of 30 and learning rate of 870.X-shift clustering analysis was performed using expression of the same 6 markers, with the k nearest neighbor value of 222 and distance metric set at Euclidean.
For each cluster in each of the two tSNE plots the MFI of each marker was determined and relative expression calculated.The proportion of cells within each cluster of the tSNE plot were assessed for each sample and the mean and standard deviation calculated.For both the CD3 + and Treg cell tSNE, each marker of interest was manually gated and applied to the tSNE plot to visualize positively labeled cells.

Figure 1 .
Figure 1.Treg cells are less abundant with reduced FOXP3 in early pregnancy failure Peripheral blood lymphocytes were assessed by flow cytometry in proven fertile (Fert, n = 15) and early pregnancy failure subjects (EPF, n = 27).The population of viable CD3 + T cells (A) was assessed for CD4 and CD8 expression to define the percentage of CD4 + CD8 À (B) and CD8 + CD4 À (C) T cells, and the CD4 + :CD8 + ratio was calculated (D).The proportion of double-negative CD4 À CD8 À (E) and double-positive CD4 + CD8 + (F).Within the CD3 + CD4 + population, FOXP3 + CD25 + CD127 -/lo Treg cells were identified (G) and quantified (H), then within the Treg cell pool the MFI of FOXP3 (I) was measured.Symbols indicate individual study participants, and the mean G SEM of each group are shown.The effect of fertility status was analyzed by t test with FDR correction to account for multiple comparisons.*p < 0.05, **p < 0.01.

Figure 2 .
Figure 2. Treg cells have an altered phenotype indicating reduced functional fitness in early pregnancy failure Peripheral blood lymphocytes were assessed by flow cytometry and CD25 + FOXP3 + CD127 -/lo Treg cells within the CD3 + CD4 + pool were analyzed in proven fertile (Fert, n = 15) and early pregnancy failure subjects (EPF, n = 27) (A).Within the Treg cell pool the percentage of Helios + cells (A) and the MFI of Helios within Helios + Treg cells (B) was measured.Helios + Treg cells as a proportion of CD4 + T cells (C).The proportion of Treg cells expressing suppressive marker CTLA4 (D) and the MFI of CTLA4 within CTLA4 + Treg cells (E).The proportion of Treg cells expressing proliferative marker Ki67 (F).The proportion of Treg cells according to degree of suppressive capacity indicated by HLADR À CD45RA + (G), HLADR À CD45RA À (H), HLADR + CD45RA À (I) and HLADR hi CD45RA À (J).Treg memory and naive phenotypes according to CCR7 and CD45RA expression (K) identifying naive (Treg naive ), central memory (Treg CM ), effector memory (Treg EM ) and terminally differentiated effector memory cells expressing CD45RA (Treg EMRA ) Treg cells.The proportion of Treg naive and Treg CM within the CD4 + population are shown in (L) and (M), respectively.For (A-J, L, M), symbols indicate individual study participants, and the mean G SEM of each group are shown.For (K), the subtypes of memory and naive Treg cells are shown as a stacked bar graph with the mean G SEM of each subgroup indicated.The effect of fertility status was analyzed by t test with FDR correction to account for multiple comparisons.*p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3 .
Figure 3. CD4 + and CD8 + T cells exhibit altered phenotypes in early pregnancy failure Peripheral blood CD4 + (A-F) and CD8 + (G-K) T cells were assessed by flow cytometry in proven fertile (Fert, n = 15) and early pregnancy failure subjects (EPF, n = 27).CD4 + T conventional (Tconv) cells were defined as CD3 + CD4 + and not FOXP3 + CD25 + CD127 -/lo and CD8 + T cells were defined as FOXP3 + CD8 + Treg cells (G) or FOXP3 À CD8 + Tconv cells (H-K).Tbet expression was measured in CD4 + Tconv (A) and CD8 + Tconv cells (H) and the ratio of CD4 + Treg cells to CD4 + Th1 cells (Treg:Th1, B) was calculated.RORgt expression was measured in CD4 + Tconv (C) and CD8 + Tconv cells (I), and the ratio of CD4 + Treg cells to CD4 + Th17 cells (Treg:Th17, D) was calculated.The percentage of proliferating CD4 + Tconv (E) and CD8 + Tconv (J) cells was evaluated by Ki67 expression.CD4 + Tconv (F) and CD8 + Tconv (K) memory and naive phenotypes were measured by assessing CCR7 and CD45RA expression to define; naive (T naive ), central memory (T CM ), effector memory (T EM ) and effector memory expressing CD45RA (T EMRA ) Treg cells.For (A-E) and (G-J), symbols indicate individual study participants, and the mean G SEM of each group are shown.For (F) and (K), memory and naive phenotypes are shown as a stacked bar graph with the mean G SEM of each subgroup.The effect of fertility status was analyzed by t test with FDR correction to account for multiple comparisons.*p < 0.05.

Figure 4 .
Figure 4. Unbiased t-distributed stochastic neighbor embedding (tSNE) and X-shift analysis of peripheral blood CD3 + T cells Peripheral blood T cells from proven fertile (Fert, n = 15) and early pregnancy failure subjects (EPF, n = 27) were analyzed by flow cytometry.A downsample of 7,344 CD3 + T cells from each sample were concatenated, visualized using the tSNE algorithm, and 16 unique cell clusters identified using X-shift analysis, which are represented by different colors (A).The relative mean fluorescence of each marker in each cluster was calculated and shown as a heatmap (B).Clusters were classified into subgroups of CD4 + Treg cells (C), CD4 + Tconv cells (D), CD8 + cells (E), and either double-negative CD4 À CD8 À or double-positive CD4 + CD8 + T cell (F) clusters, and the proportion of each cluster for each participant group calculated and shown as the mean G SEM.The effect of fertility status was determined by t test.*p < 0.05.

Figure 5 .
Figure 5. Unbiased t-distributed stochastic neighbor embedding (tSNE) and X-shift analysis of peripheral blood Treg cells Peripheral blood T cells from proven fertile (Fert, n = 15) and early pregnancy failure subjects (EPF, n = 27) were analyzed by flow cytometry.A downsample of 418 Treg (CD3 + CD4 + FOXP3 + CD25 + CD127 -/lo ) cells from each sample were concatenated, visualized using the tSNE algorithm, and 8 unique cell clusters identified using X-shift analysis, which are represented by different colors (A).The relative mean fluorescence of each marker in each cluster was calculated and shown as a heatmap (B).The proportion of each cluster for each Fert and EPF subject was calculated and shown as mean G SEM (C), with the effect of fertility status determined by t test, *p < 0.05.The relative expression of FOXP3 and CD25 are shown on the tSNE plot as a heatmap (D) and Treg cells were manually gated for positive expression of CCR7, CD45RA, HLADR, Helios, CTLA4, and Ki67, with each marker then mapped onto the tSNE plot (E).

Figure 6 .
Figure 6.RNA-seq reveals altered transcriptome in Tconv cells and Treg cells in early pregnancy failure Tconv and Treg cells isolated from cryopreserved PBMCs from proven fertile and EPF subjects by FACS were stimulated in vitro for 48 h, before RNA was isolated and used to construct cDNA libraries for RNA-seq on an Illumina HiSeq platform (A).A principal-component analysis (PCA) plot for all Tconv and Treg cell RNAseq samples, with ellipses showing the 95% CI for Tconv and Treg cell clustering around the mean (B).PCA plot shows Tconv cell samples from proven fertile (n = 11) and EPF subjects (n = 12) (C) and total differentially expressed genes identified in Tconv cells from EPF versus proven fertile subjects with an FDR-adjusted p < 0.05 are displayed in a Venn diagram (D).PCA plot shows Treg cell samples from proven fertile (n = 6) and EPF subjects (n = 8), after RUVg normalization (E).Total differentially expressed genes identified in Treg cells from EPF versus proven fertile subjects with FDR-adjusted p < 0.05 are displayed in a Venn diagram (F).

Figure 7 .
Figure 7. Differential expression of cytokines and immune-regulatory genes in Treg cells in early pregnancy failure A volcano plot displaying differentially expressed genes (DEGs) identified in Treg cells from proven fertile (n = 6) and EPF subjects (n = 8) was constructed (A).Significantly differentially expressed genes that are upregulated in EPF Treg cells are red, whereas downregulated genes are blue and the top 20 with the lowest adjusted p-values are labeled.Genes outside the p value cutoff for significance (Bonferroni-adjusted p < 0.05) are in gray.A GO network plot showing enriched Molecular Function terms and the associated differentially expressed genes for Treg cells from EPF compared with proven fertile subjects was constructed (B).The size of the dots for GO terms is larger when it is associated with more differentially expressed genes.The color of the dots indicates if the gene is upregulated (red) or downregulated (blue) in EPF Treg cells.Differentially expressed genes in Treg cells from proven fertile and EPF subjects that encode cytokines, cytokine receptors, chemokines, chemokine receptors, transcription factors, and other immune-regulatory molecules are displayed in a heatmap (C) showing relative expression in each sample based on Z scores calculated from the logCPM values.Red indicates the gene is upregulated relative to mean expression across all samples; blue indicates downregulation.*DEGs identified by in silico analysis to be targeted by FOXP3 transcription factor regulation are marked with asterisks.# Individuals in the EPF group classified as positive for autoimmune status (see TableS2) are identified by hash symbol.

Figure 8 .
Figure 8. Impact of autoimmune status on Treg cell phenotype in early pregnancy failure The autoimmune status of 25/27 EPF subjects was designated ''Yes'' or ''No'' according to ESHRE clinical guidelines (see TableS2).Data on autoimmune status is not available (n/a) for proven fertile women or 2/27 EPF women.Values for flow cytometry parameters from proven fertile and EPF subjects are displayed in a heatmap showing relative score in each sample based on Z scores calculated from data shown in Figures1, 2, and 3. Higher intensity of red indicates the parameter is higher relative to mean values across all samples.See FigureS9for additional visualization of flow cytometry data according to autoimmune status.